Shining a light on UV-fluorescent floral nectar after 50 years

Nature is aglow with numerous captivating examples of UV-fluorescence in the animal kingdom. Despite a putative role as a visual signal, exploration of UV-fluorescence in plants and its role in plant-animal interactions is lagging in comparison. Almost 50 years ago, UV-fluorescence of floral nectar, a crucial reward for pollinators, was reported for 23 flowering plant species. Since this intriguing discovery, UV-fluorescent nectar has only seldom been addressed in the scientific literature and has not been scrutinized in a phylogenetic or ecological context. Here, we report the prevalence of vibrant UV-fluorescent floral nectar across the family Cleomaceae, including the first photographic documentation in vivo colour for flowering plants. Though Cleomaceae flowers are morphologically diverse varying in colour, nectary prominence, and nectar volume, UV-fluorescent floral nectar may be a ubiquitous characteristic of the family. Fluorescence spectra show that the identity and number of fluorescent compounds in floral nectar may differ among Cleomaceae species. As Cleomaceae pollinators range from insects to bats and birds, we suggest that the UV-fluorescent floral nectar not only functions as a visual cue for the diurnal pollinators but also for the nocturnal/crepuscular pollinators in low light settings.

From vibrant colours to striking patterns, flowering plants display an astonishing array of characteristics that act as visual signals for pollinators 1 .The innate and learned preferences of pollinators to these suites of visual cues encourage visitation 2,3 , with flowering plants commonly offering rewards such as nectar in exchange for pollen transfer.Given the intricate ties among pollinators and floral displays, flower colour research is key to shedding light on plant-pollinator interactions and the evolution and diversity of floral form.Ultraviolet (UV)fluorescence, a floral feature that may function as a visual signal for pollinator attraction, represents a significant gap in our knowledge of flower colour.
UV-fluorescence is a type of luminescence in which UV radiation is absorbed and longer wavelength light is emitted.Unlike the well-known UV nectar guides (i.e., patterns of UV-absorbance and -reflectance) that can only be perceived by animals with UV-receptors (e.g., insects and birds) 1,4 , the lower energy light emitted via UV-fluorescence can occur in the spectral range visible to humans (and many other animals) 1,5 .Further, UV-fluorescence has not been studied as extensively as UV nectar guides 1,[6][7][8] .However, UV-fluorescence is of growing interest with many recent discoveries of this phenomenon across the animal kingdom (e.g., the hair of nocturnal springhare and flying squirrels 9,10 , skin of salamanders and catsharks 11,12 , and the bones of toadlets and chameleons 13,14 ).Behavioural studies within the animal kingdom suggest that UV-fluorescence may be more than a coincidental by-product of chemical structure [15][16][17] .For example, female jumping spiders (Cosmophasis umbratica, Salticidae) have appendages that fluoresce bright green under UV radiation; in the absence of UV radiation, male jumping spiders do not perform typical courtship behaviour with non-fluorescing females 17 .Similarly, budgerigars (Melopsittacus undulatus, Psittacidae) have UV-fluorescent yellow plumage on their crown and cheeks; both sexes prefer budgerigars of the opposite sex with fluorescent plumage over those with masked fluorescence (i.e., concealed with UV-absorbing chemicals) 15 .Despite numerous captivating examples and a putative function as a visual signal in animals, the prevalence and significance of UV-fluorescence across flowering plants has scarcely been investigated.
Nearly 50 years ago, Thorp et al. 18 were among the first to report the brilliant UV-fluorescence of nectar in flowering plants and suggested that this phenomenon functions as a visual signal for bees.Out of the 102 flowering plant species examined, 23 had nectar that fluoresced yellow to blue with varying degrees of intensity and the majority pollinated by bees 18 .Apart from nectar fluorophore (i.e., fluorescent molecule) identification for three species [19][20][21] , our understanding of UV-fluorescent nectar has seen limited progress since its discovery.Suggestions that nectar fluorescence could be used as an indicator of secretory cells in vivo 22,23 and conceptual arguments about its ecological importance 24,25 have not significantly enriched our fundamental knowledge of UV-fluorescent nectar.Yet, the presence of UV-fluorescence was recently reported in the prey traps of several www.nature.com/scientificreports/carnivorous plant species and in the anthers and pollen of numerous flowering plants species [26][27][28] .Like the UV-fluorescent animal studies, behavioural experiments with UV-fluorescent prey traps and an anther/pollen fluorophore show that this phenomenon plays a role in animal attraction 26,27 .Pitcher plants (Nepenthes khasiana, Nepenthaceae) with masked fluorescence catch significantly less insect prey 26 and bees are attracted to filter paper containing a fluorescent compound identified from anthers and pollen 27 .Though UV-fluorescence presumably contributes to the array of visual cues involved in pollinator attraction, further exploration is needed to determine the prevalence of UV-fluorescent nectar across flowering plants, characterize its molecular basis, and to establish a link to pollinator interactions.
Though UV-fluorescence in nature is a fascinating phenomenon to observe, to the best of our knowledge, there are only four published photographs of UV-fluorescent nectar 18,29,30 (Fig. 1).Unfortunately, the photographs are monochromatic, captured ex vivo or marred by blurriness, and fail to ignite curiosity parallel to that of UVfluorescence research in the animal kingdom.Here, we present the first in vivo colour images of UV-fluorescent nectar in flowering plants.We show UV-fluorescent nectar across Cleomaceae, a relatively small family with morphologically diverse flowers and a broad range of diurnal and nocturnal/crepuscular pollinators 31,32 .Moreover, we discuss the current state of flower florescence research, including the potential phylogenetic and ecological implications of UV-fluorescent floral nectar.

Plant material
The

Nectar volume measurements
Nectar was extracted from flowers with microcapillary tubes (0.4 mm i.d., 75 mm length; Drummond Scientific, Broomall, PA, USA) between 10:00 and 12:00.The height of nectar drawn into the microcapillary tube was measured to the nearest 0.5 mm and used to calculate nectar volume.For species with nectar too viscous to draw into a microcapillary tube (i.e., M. giganteus and T. houtteana) the following protocol was used: the flower was removed from the plant at the base of the pedicel, water was added to the nectar with a pipette, the pipette tip was used to manually swirl the nectar solution without aspirating, the nectar solution was extracted from the nectary with microcapillary tubes, and the amount of added water was subtracted from the total nectar solution volume.For each Cleomaceae species, nectar was collected from a minimum of three plants (i.e., biological replicates) and 1-30 flowers per plant.The five species with the highest nectar volumes were selected for in vivo photography and fluorescence spectra analysis.

Literature review
A literature review was conducted using Google Scholar to determine the phylogenetic distribution and possible ecological implications of UV-fluorescent nectar.As Thorp et al. 18 is the key article that brought awareness to UV-fluorescent nectar, scientific literature citing this work was examined for mentions of additional species exhibiting this phenomenon and evaluations of its function.Further, we extended our review by searching for scientific literature containing the terms "ultraviolet", "fluorescent", and "nectar".Studies that examined nectar for fluorescence after extraction from a pollinator or introduction of a solvent or stain were excluded due to the possibility of nectar modification.Examples of UV-fluorescence in other plant features were identified through review of the abovementioned literature.All methods were performed in compliance with the relevant guidelines and legislation.

Results
For the nine Cleomaceae species examined here, nectar is secreted by receptacular nectaries located between the perianth and stamens, or perianth and androgynophore (i.e., stalk-like structure subtending the reproductive organs; e.g., G. gynandra) 32,34 .The nectaries are diverse in form, ranging from adaxial protrusions or concavities to annular disks, and from inconspicuous to a prominent component of the flower 32 .The average nectar volume secreted varied from 0.01 to 19.73 µL with the highest average nectar volumes (> 0.5 µL) secreted by M. giganteus, T. houtteana, P. dodecandra, S. hirta, and C. violacea and lowest (< 0.5 µL) by S. rutidosperma, C. amblyocarpa, S. monophylla, and A. viscosa (in descending order; Table 1).Of note, Lunau et al. 35 mentioned S. monophylla as an example of a species with a false nectary (i.e., a glossy surface mimicking nectar); however, we show that its nectary is functional.Arivela viscosa was excluded from subsequent fluorescence analyses due to insufficient nectar volumes.For the remaining nine Cleomaceae species, the floral nectar is colourless under white light but exhibits vibrant blue fluorescence when illuminated by UV-A radiation with peak intensity at 365 nm (Fig. 2).
With nectaries and nectar that are not obscured by the perianth or stamens, C. violacea, P. dodecandra, and T. houtteana have the most visually striking examples of UV-fluorescent nectar.Under white light, the nectar of C. violacea is challenging to discern from the green three-lobed nectary (Fig. 2a).Yet, when excited with UV-A radiation, the vividly fluorescent nectar droplets are easily distinguished from the nectary and contrast the less intense red fluorescence of chlorophyll 36 (Fig. 2b,c).Similarly, under white light, the nectar of P. dodecandra and T. houtteana accumulates on top of an orange cup-shaped nectary and light green 'V'-shaped nectary, respectively (Fig. 3a,c).Under UV-A radiation, the nectar of both species intensely fluoresces (Figs.2d, 3b,d).In addition, the vasculature within the petals of P. dodecandra fluoresces blue and the petals of T. houtteana fluoresce bright pink.www.nature.com/scientificreports/Cleomaceae species with nectaries and nectar partially or entirely concealed by the perianth and stamens include C. amblyocarpa, M. giganteus, S. hirta, S. monophylla, and S. rutidosperma.For instance, the perianth of S. hirta and M. giganteus partially obscure the adaxially secreted nectar (Fig. 4a,c).Though both species secrete UV-fluorescent nectar (Fig. 2d), the blue fluorescence of the nectar does not appear as vibrant against the intense fluorescence of the other floral structures (Fig. 4b,c).As C. amblyocarpa, G. gynandra, S. monophylla, and S. rutidosperma have obscured nectaries and/or low nectar volumes (< 0.5 µL), nectar was pooled from multiple flowers and observed in microcapillary tubes.Whether exposed or obscured, the nectar of the nine Cleomaceae species fluoresces blue under UV-A radiation (Fig. 2d, Supplementary Fig. S1).The intensity of fluorescence varies between and within genera and can differ within species (Supplementary Fig. S1).Of note, the pollen fluorescence can also be rather vivid though the nectar fluorescence often steals the show (i.e., P. dodecandra) (Fig. 3b).Like the Cleomaceae taxa, B. rapa has receptacular nectaries between the perianth and stamens and UV-fluorescent nectar (Supplementary Fig. S1).
For the five species that secreted the greatest volumes of nectar, the nectar fluorescence excitation maxima ranged from 309 to 396 nm and the emission maxima varied from 416 to 476 nm (Fig. 5, Supplementary Table S2).The nectar fluorescence spectra of C. violacea, M. giganteus, and T. houtteana each had one set of emission and excitation peaks, while P. dodecandra and S. hirta had two sets of peaks.No two species had the same nectar fluorescence spectra.The Cleomaceae nectar fluorescence spectra maxima did not correspond to that of genistein (Supplementary Table S2), which fluoresced green instead of blue, but more closely resembled the fluorescence spectra of hydroxycinnamate derivatives 27 .

Evolution of UV-fluorescent nectar
Though there is limited evidence about its prevalence, the scattered distribution of UV-fluorescent nectar suggests that this phenomenon arose multiple times across flowering plants (Fig. 6).Including the taxa from our investigation, UV-fluorescent nectar has been documented for 41 species (19 families), predominantly belonging to the eudicots 18,21,22,30,37 (Table 2).For most of these families, the occurrence of UV-fluorescent nectar varies within family, genus, or even species (Table 2; Supplementary Table S3).For instance, Thorp et al. 18 noted the presence of UV-fluorescent nectar in five Prunus (Rosaceae) species and absence in four, including UV-fluorescent nectar in peach but not nectarine flowers (i.e., varieties of P. persica).The variability between closely related individuals suggests that the genetic mechanisms governing fluorophore biosynthesis are conserved but differentially expressed within lineages.While nectar fluorescence is variable in Brassicaceae (sister family to Cleomaceae), this phenomenon may be a unifying feature of Cleomaceae as all ten species investigated, which span multiple genera and clades, exhibit nectar fluorescence to varying degrees of intensity 18 (Supplementary Fig. S1).
With the possibility of multiple independent origins of UV-fluorescent nectar, the question arises: are distinct fluorophores responsible for nectar fluorescence throughout the flowering plant phylogeny?Like nectar, species with anthers and pollen that emit blue fluorescence under UV radiation are dispersed across flowering plants 27,28 .Mori et al. 27 identified the anther and pollen fluorophores of five species in four different eudicot families as hydroxycinnamate derivatives and suggested their widespread distribution and shared biosynthetic pathway for taxa with fluorescent blue anthers and pollen.Alternatively, UV-fluorescent compounds can be unique to specific clades and thus act as taxonomically informative characters.For example, ester-linked ferulic acid exclusively occurs in the cell walls of the monophyletic clades commelinid monocots and core Caryophyllales 38,39 .When observed with UV-fluorescence microscopy, cell walls with ester-linked ferulic acid fluoresce blue when in water and green when in acid 38 .For the five Cleomaceae species examined, the distinct fluorescence spectra suggest that different compound(s) are responsible for the nectar fluorescence in each species.Further, the two sets of Though often perceived as a simple sugar solution, nectar consists of a complex array of biomolecules and microorganisms (i.e., bacteria and fungi) 34,40,41 .Major constituents such as water, carbohydrates, and amino acids reward pollinators; proteins can tailor nectar chemistry for pollinators and prevent microbial infections; and secondary metabolites including scented and coloured compounds can contribute to pollinator attraction [40][41][42][43][44] .Since biomolecules, including large macromolecules such as proteins, can act as fluorophores and microorganisms can contain fluorophores 36 , the complexity of nectar poses a challenge for the identification of UVfluorescent components.Yet, in response to the fascinating finding of Thorp et al. 18 , Scogin [19][20][21] identified the UV-fluorescent compounds in the nectar of three Malvaceae species as a genistein-related isoflavone and its glucoside (Fremontodendron californicum and F. mexicanum) and a hydroxycoumarin (Bombax ceiba).Since this discovery, there have been several reports of a hydroxycinnamate derivative (i.e., chlorogenic acid; pollen/ anther fluorophore 27 ), genistein-related isoflavone and glucosides, and a hydroxycoumarin (i.e., aesculetin) in the nectar of diverse taxa, including two species with known UV-fluorescent nectar 18,[45][46][47][48][49][50] (Robinia pseudoacacia, Fabaceae and Fagopyrum esculentum, Polygonaceae; Supplementary Table S4).However, these studies did not examine the nectar samples for UV-fluorescence.

Potential ecological function of fluorescent nectar
This seemingly prevalent phenomenon in Cleomaceae brings forth the question: does UV-fluorescent nectar serve as a visual cue for the array of Cleomaceae pollinators?The significance of UV-fluorescence for pollinator attraction has been debated.Thorp et al. 18 posited that UV-fluorescent nectar functions as a visual signal for foraging bees.However, this hypothesis has been criticized due to concerns that the emitted fluorescence may be imperceptible to insects amid the reflected light 24,25 .While describing the colour of a flower may appear straightforward, providing an ecologically relevant description proves challenging as colour perception is dependent on the sensory and processing capabilities of the observer 1 .For instance, insects and birds have UV photoreceptors, while humans do not 1,4 .As a result, the perceived colour of a flower may drastically differ between humans and www.nature.com/scientificreports/pollinators.Under sunlight, nectar fluorescence may not be conspicuous to the human eye, yet behavioural assays have shown that honeybees make fine colour discriminations and are attracted to a UV-fluorescent compound (i.e., chlorogenic acid) 27,51 .Further, honeybees have a blue photoreceptor maximally activated at 436 nm, lying within the range of Cleomaceae nectar fluorescence emission maxima (416-476 nm; Fig. 5, Supplementary Table S2), and an innate preference for blue colours 52,53 .Unlike Thorp et al. 18 that reported bees as the primary pollinators of taxa with UV-fluorescent nectar, Cleomaceae consists of both generalist species pollinated by a variety of insects and sometimes hummingbirds (e.g., A. viscosa, Cleomella arborea, P. dodecandra) [54][55][56] and specialist species solely pollinated by bats (e.g., M. giganteus and T. houtteana) 57,58 .Though echolocation and olfaction play important roles in bat orientation and foraging 59,60 , all bat species have functional eyes 61 .For example, one of the bat pollinators of T. houtteana (i.e., Glossophaga soricine, Phyllostomidae) is colour-blind but able to perceive UV radiation as well as some human-visible light 61 .Further, Domingos-Melo et al. 62 suggested that bats are attracted to the brightness of visible light reflecting off chiropterophilous (i.e., bat-pollinated) flowers at dusk.In addition to bats, nocturnal/crepuscular pollinators of Cleomaceae also include hawkmoths (e.g., G. gynandra) 63 .Investigations on animals show that UV-fluorescence is most prevalent and intense for nocturnal species and suggest that this phenomenon is an overlooked visual signal for nocturnal animals 9,10,[64][65][66] .Likewise, a behavioural assay involving the UV-fluorescent pitcher plant N. khasiana revealed that UV-fluorescence may play a role in insect attraction in low light settings 26 .Prey capture of unmasked pitcher plants primarily occurred at night and masking of the fluorescent blue rim significantly reduced the capture of insect prey 26 .With evidence from the behavioral assays suggesting fluorescence aids in the attraction of both diurnal and nocturnal/crepuscular pollinators 26,27 , it is possible that fluorescent nectar in Cleomaceae not only acts as a visual signal for daytime pollinators but may also assist in the attraction of  A box is present for orders that have data for at least one species.The phylogeny is adapted from that of the Angiosperm Phylogeny Group 67 and data is summarized from Thorp et al. 18 , Scogin 21 , Roshchina et al. 22 , Nakanishi 30 , Davis et al. 37 , and the present study.
Table 2. Flowering plant species with UV-fluorescent floral nectar.Adapted from the data of Thorp et al. 18 , Scogin 21 , Roshchina et al. 22 , Nakanishi 30 , Davis et al. 37 , and the present study (indicated by an asterisks).

Figure 1 .
Figure 1.UV-fluorescent floral nectar of Prunus species.(a) A spot of P. amygdalus nectar on filter paper under long-and short-wavelength UV radiation.From Thorp et al. 18 .Reprinted with permission from AAAS.(b) A P. persica flower with nectar under UV radiation.From Radice and Galati 29 .Reprinted with permission from SNCSC.n, nectar.Scale bar, 2.5 mm (b).

Figure 4 .
Figure 4. UV-fluorescent floral nectar of Sieruela hirta and Melidiscus giganteus.(a, b) Sieruela hirta under white light (a) and UV-A radiation (b).(c, d) Melidiscus giganteus under white light (c) and UV-A radiation (d).Arrowheads are pointing to the partially exposed nectar.Scale bars, 1 cm.

Figure 6 .
Figure 6.Phylogenetic distribution of UV-fluorescent and non-fluorescent floral nectar across flowering plants.A box is present for orders that have data for at least one species.The phylogeny is adapted from that of the Angiosperm Phylogeny Group 67 and data is summarized from Thorp et al.18 , Scogin21 , Roshchina et al.22 , Nakanishi30 , Davis et al.37 , and the present study.